与大劣按蚊先天免疫相关丝氨酸蛋白酶的克隆及分析

目的克隆及分析与先天免疫相关的大劣按蚊差异基因。方法利用抑制性消减杂交 （ suppression subtractive hybridization, SSH）方法克隆大劣按蚊差异基因，并对与先天免疫相关的差异基因（文中命名为T6基因）进行生物信息学分析及半定量实验，分析约氏疟原虫感染前后其转录的变化，探讨它与按蚊抗疟原虫感染的先天免疫反应的关系。结果目前克隆的差异基因包含以下几类：①与先天免疫相关的酶类；②与能量代谢有关的酶：③与信号传递和调节有关的因子。对与先天免疫相关的分子进行的半定量实验表明，大劣按蚊T6基因的转录与感染疟原虫相关。结论本研究成功地克隆了与先天免疫相关的大劣按蚊差异基因，进一步的实验分析提示该基因可能与按蚊抗约氏疟原虫感染的黑化反应相关。     

Resistance of Anopheles gambiae sensu lato to Pirimiphos-methyl Insecticide in Kakamega County,Highlands of Western Kenya

BackgroundInsecticide treated bed nets and Indoor residual spraying remains the principal interventional malaria control strategies. To achieve malaria disease eradication, vector control programmes that monitor insecticide resistance profiles are necessary.ObjectiveThe study evaluated pirimiphos-methyl susceptibility of Anopheles gambiae sensu lato in Kakamega County, western Kenya.MethodsAdult Anopheles gambiae sensu lato mosquitoes were assayed using World Health Organization tube bioassay against 0.25% pirimiphos-methyl. Susceptible and non-susceptible populations were characterized to species-level using Polymerase Chain Reaction. Susceptible and resistant mosquitoes were further subjected to G119S Acetylcholisterase (ace 1R) mutation detection.ResultsAnopheles arabiensis was the predominant species in all study population Mumias east (62%), Malava (68%), Ikolomani (77%) and Lurambi (82%). Results showed phenotypic susceptibility to pirimiphos-methyl. Mortality was low in Mumias east (80.6%) and high in Lurambi (89.0%). G119S mutations ranged from 3.0% to 8.9% in Anopheles arabiensis whereas G119S mutations were relatively low ranging from 0.0% to 3.1% in Anopheles gambiae s.s populations. Study populations tested were consistent with Hardy-Weinberg equilibrium (P>0.05).ConclusionWe observed pirimiphos-methyl resistance in Anopheles arabiensis and Anopheles gambiae s.s. study populations. Results showed G119S mutation in resistance population. Resistance monitoring and management are urgently required.     

Induction of mosquito hemolymph proteins in response to immune challenge and wounding

The rapid induction of proteins in the hemolymph of the mosquito, Anopheles gambiae, was examined after wounding or injection of immune elicitors (Escherichia coli, lipopolysaccharide, laminarin, zymosan). One-dimensional gel electrophoresis revealed at least six hemolymph polypeptides >25 kDa that consistently appeared after any breech of the cuticle. All of these polypeptides appeared in the hemolymph within 30 min and reached a maximum concentration after approx. 6 h. No proteins were specifically induced by bacteria or bacterial or fungal cell wall products, however two constitutively expressed proteins were repressed by these injections. Patterns of hemolymph proteins were further analyzed by two-dimensional electrophoresis. Seven spots were enhanced or induced 2 h after injection in four replicate experiments. An additional two spots demonstrated some variability between replicates, but were generally responsive to injection. These rapidly induced polypeptides are candidates for regulating and initiating the mosquito’s responses to pathogens and wounding.     

The Role of Serpin and Cystatin Antiproteases in Mucosal Innate Immunity and their Defense against HIV

Antiproteases play diverse roles in nature, from regulating protease activity to innate defense against microorganisms. Recently, antiproteases have been shown to play important roles in HIV pathogenesis including, inhibiting HIV binding and replication and reducing activation and inflammation of susceptible cells. They have also been implicated as one of the initial host responders, in plasma, to control replication of HIV. More recently, antiproteases expressed at the mucosal surface have been linked to reduced susceptibility to HIV infection in HIV‐exposed sero‐negative individuals. These factors are expressed in the epithelial layer of the female genital tract, thus at the frontline of defense against mucosal infection. This review focuses on the specific antimicrobial roles of antiproteases, focusing on serpins and cystatins, with an emphasis on their known and potential roles in HIV infection. Their potential as therapeutic interventions to combat HIV is also discussed.     

云南微小按蚊饲养观察及其人工感染间日疟原虫初步观察

为了建立实验室云南微小按蚊饲养品系,以观察该蚊生长发育情况及其人工感染间日疟原虫的敏感性,采用常规人工方法饲养微小按蚊和直接叮咬吸血法进行人工感染间日疟原虫观察.实验表明微小按蚊在室温26～28℃、相对湿度60%～75%、水温23～25℃,每日光照12 h的实验室条件下,其孵化率、化蛹率、羽化率分别为83.1%、78.2%、92.6%,并观察10批次微小按蚊,他们间的孵化率、化蛹率及羽化率无统计学差异;人工感染6例间日疟患者中,3例感染者微小按蚊蚊胃卵囊阳性,它们的卵囊感染率分别为61.6%、87.5%和100.0%.结果表明云南微小按蚊饲养条件在26～28℃、相对湿度60%～75%、水温23～25℃下生长发育较好,同时,该蚊对间日疟原虫仍然敏感.     

Hemolymph proteins of Anopheles gambiae larvae infected by Escherichia coli

Anopheles gambiae is a major vector of human malaria and its immune system in part determines the fate of ingested parasites. Proteins, hemocytes and fat body in hemolymph are critical components of this system, mediating both humoral and cellular defenses. Here we assessed differences in the hemolymph proteomes of water- and E. coli-pricked mosquito larvae by a gel-LC-MS approach. Among the 1756 proteins identified, 603 contained a signal peptide but accounted for two-third of the total protein amount on the quantitative basis. The sequence homology search indicated that 233 of the 1756 may be related to defense. In general, we did not detect substantial differences between the control and induced plasma samples in terms of protein numbers or levels. Protein distributions in the gel slices suggested post-translational modifications (e.g. proteolysis) and formation of serpin-protease complexes and high Mr immune complexes. Based on the twenty-five most abundant proteins, we further suggest that major functions of the larval hemolymph are storage, transport, and immunity. In summary, this study provided first data on constitution, levels, and possible functions of hemolymph proteins in the mosquito larvae, reflecting complex changes occurring in the fight against E. coli infection.